Subcellular dynamics of leghemoglobin is modulated by its site-specific serine phosphorylation during symbiotic nitrogen fixation in Lotus japonicus

Symbiotic nitrogen fixation (SNF) relies on aerobic respiration, yet the key enzyme, nitrogenase, is extremely oxygen labile. Leghemoglobin (Lb) resolves this "oxygen paradox" by buffering and facilitating O2 transport. However, the dynamic regulation of Lb during nodule development remains poorly understood. Earlier results from our laboratory demonstrated that site-specific serine phosphorylation of Lb reduces its oxygen sequestration capacity. Here, we investigated the spatio-temporal regulation of Lb with the progress of rhizobial load during SNF. Fluorescence immunohistochemistry (FIHC) using anti-Lb antibody revealed that its localization gradually shifted from the plasma membrane to the cytoplasm of infected cells as nodules mature. Using phospho-peptide (Lb) specific antibodies, we found that serine phosphorylation triggers this translocation. Furthermore, FIHC in conjunction with immunoprecipitation followed by immunoblotting with phospho- and non-phospho-peptide specific antibodies demonstrated that the non-phosphorylated form is detectable as early as 9 dpi, whereas the phosphorylated forms were first detected at 11 dpi and progressively accumulated during nodule maturation. This spatio-temporal transition coincides with increasing rhizobial colonization and is accompanied by a decline in the non-phosphorylated pool. Therefore, the increased cytoplasmic pool of phosphorylated Lb, which exhibits reduced oxygen sequestration capacity, likely functions in promoting oxygen transport to sustain elevated rhizobial respiration. Together, these findings demonstrate that site-specific serine phosphorylation represents one of the key regulatory mechanisms linking Lb localization dynamics with progression of rhizobial infection, thereby contributing to the maintenance of oxygen homeostasis during SNF.